An estimated $1.8 billion dollars is spent for each new successful drug developed, primarily in failed clinical trials due to lack of efficacy and safety. New approaches for rapid identification and early preclinical validation of novel therapeutic targets are crucial to make important go/no-go decisions and curb the cost of developing new cancer treatments. For decades, genetically engineered mouse models have provided a powerful platform to study disease initiation and maintenance, the tumor microenvironment and the responsiveness of cancers to known or novel therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models with various cancer predisposing gene combinations have limited their practical utility in the drug discovery process. RNA interference (RNAi), a mechanism that controls gene expression, can be exploited experimentally to silence nearly any gene target. By expressing synthetic short hairpin RNAs (shRNAs) in mice, RNAi serves as a fast alterative to gene deletion. In theory, RNAi- GEMMs are a powerful platform to validate candidate cancer genes and drug targets, however, using traditional approaches, up to 2 years of extensive intercrossing would be required to produce experimental cohorts, thus preventing their routine use. Hypothesis: We hypothesize that RNAi-GEMMs of cancer can be developed rapidly and cost-effectively using our embryonic stem cell (ESC) rederivation method coupled with new genome editing technologies (TALENs/CRISPRs) to introduce additional sensitizing lesions and recombinase-mediated cassette exchange (RMCE) for precise integration of tetracycline inducible shRNAs to silence specific gene targets. Preliminary data: We have previously used this technique to generate entire experimental cohorts of functional mouse models of cancer without any breeding. Specific Aims: This project entails preclinical testing of RNAi-mediated Ptgs2 (Cox-2) inhibition in a new mouse model of Familial Adenomatous Polyposis (FAP) and determining its ability to model therapeutic administration of the Cox-2 inhibitor, Celecoxib, which reduces disease in mice and is the FDA approved treatment for FAP. In Specific Aim #1, we will use the CRISPR/Cas9 system to create a truncating mutation in the Apc tumor suppressor gene in newly derived ESCs, monitor the mice generated by blood sampling and macroscopically examine their intestines for polyp formation to confirm their ability to phenocopy the APCMin mouse model and mirror the human condition. In Specific Aim #2, we will introduce an shRNA targeting Cox-2 and compare its effects to the FDA approved therapy, Celecoxib, to further assess the therapeutic potential of our ESC-derived RNAi- GEMMs. Together, these studies will define a new paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAi-GEMMs that will serve as innovative research tools, guiding the development of novel and effective therapeutics.